If you wanted to know if your child had a fever or be certain that the roast
in the oven was thoroughly cooked, you would, of course, use a thermometer that
you trusted to give accurate readings at any temperature within its range. However,
it isn't that simple for researchers who need to measure temperatures
in microfluidic systems-tiny, channel-lined devices used in medical diagnostics,
DNA forensics and "lab-on-a-chip" chemical analyzers-as their
current "thermometer" can only be precisely calibrated for one reference
temperature. Now, researchers at the National
Institute of Standards and Technology (NIST) have proposed a mathematical
solution that enables researchers to calibrate the "thermometer"
for microfluidic systems so that all temperatures are covered.

Reactions taking place in microfluidic systems often require heating, meaning
that users must accurately monitor temperature changes in fluid volumes ranging
from a few microliters (a droplet approximately 1 millimeter in diameter) to
sub-nanoliters (a droplet approximately 1/10 of millimeter in diameter). A common
DNA analysis technique, for example, depends heavily on precise temperature
cycling. Ordinary thermometers or other temperature probes are useless at such
tiny dimensions, so some groups have turned to temperature-sensitive fluorescent
dyes, particularly rhodamine B. The intensity of the dye's fluorescence
decreases with increasing temperature. The idea is that the dye can be used
as a noninvasive way to map the range of temperatures occurring within a microfluidic
system during heating and, in turn, provide a means of calibrating that system
for experiments.

However, the technique currently requires the user to base all readings on
the fluorescence at a single reference temperature. Previous groups have developed
"calibration curves" that relate temperature to rhodmaine B fluorescent
intensity based on a reference temperature of about 23 degrees Celsius (a technique
first proposed by NIST researchers David Ross, Michael Gaitan and Laurie Locascio
in 2001*). But it turns out that the curves are only good for that one temperature.
In an upcoming paper in Analytical Chemistry**, the NIST team-Jayna J.
Shah, Michael Gaitan and Jon Geist-reports that changing the reference
point, such as the higher temperature when a microfluidic system is first heated,
introduces errors when a dye intensity-to-temperature calculation is done using
current methods.

"Our analysis shows that a simple linear correction for a 40 degrees
Celsius reference temperature identified errors between minus 3 to 8 degrees
Celsius for three previously published sets of calibration equations derived
at approximately 23 degrees Celsius," says lead researcher Shah.

To address the problem, the NIST team developed mathematical methods to correct
for the shift experienced when the reference temperature changes. This allowed
the researchers to create generalized calibration equations that can be applied
to any reference temperature.

Microfluidic DNA amplification (production of numerous copies of DNA from a
tiny sample) by the polymerase chain reaction (PCR) is one procedure that could
benefit from the new NIST calculations, Shah says. "PCR requires a microfluidic
device to be cycled through temperatures at three different zones starting around
65 degrees Celsius, so a useful dye intensity-to-temperature ratio would have
to be based on that temperature and not a reference point of 23 degrees Celsius,"
she explains.

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